This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Due to their high rates of mutation, large population sizes, and rapid rates of replication, RNA virus populations exist as a swarm of mutant genomes, or "quasispecies". The diversity of a quasispecies influences viral adaptation, disease severity, and response to antiviral therapies. Identifying the mechanisms that shape quasispecies diversity is necessary to better prevent virus emergence and control virus disease, particularly for arthropod-borne viruses, all of which are RNA viruses and many of which are significant emerging threats to global public health. The recent discovery of RNA interference (RNAi), the targeted destruction of RNA with high homology to a double-stranded RNA trigger, has revealed the first general pathway by which arthropods regulate viral replication. The proposed research will test the hypothesis that the RNAi response of vectors imposes selection for mutations that enable virus populations to escape RNAi, and RNAi thereby drives diversification of viral quasispecies. To test the prediction that decreasing RNAi activity should result in decreased quasispecies diversity, we will infect sham-treated Drosophila cells and Drosophila cells in which RNAi has been specifically downregulated with mosquito-borne dengue virus. Diversity of the resulting virus progeny will be determined using pyrosequencing. A similar experiment will then be conducted in live Drosophila by infecting wild type flies or mutant flies that lack specific components of the RNAi pathway with dengue virus and sequencing the resulting viral progeny.